Fluorescence detection of poison oak oil

ABSTRACT

The invention herein disclosed provides for compositions, methods for synthesizing said compositions, and methods for using said compositions, wherein the compositions and methods may be used to bind to and/or deactivate a poison oak oil, such as urushiol. The compositions and methods can be used to treat and/or reduce an inflammatory reaction and/or hypersensitivity to natural compounds found in poison oak, poison ivy, poison sumac, mango, lac tree, and cashew nut.

This application is a Continuation patent application of copending U.S.application Ser. No. 13/714,899, filed 14 Dec. 2012, which was aContinuation U.S. patent application Ser. No. 12/736,814, filed 12 Nov.2010.

RELATIONSHIP TO OTHER APPLICATIONS

This application claims priority to and benefits of the following: U.S.Provisional Patent Application No. 60/127,588, filed 13 May 2008,entitled “Fluorescence Detection And Deactivation Of Poison Oak Oil”,International Patent Application number PCT/US2009/002958, filed 13 May2009, entitled “Fluorescence Detection And Deactivation Of Poison OakOil”, U.S. National Phase patent application Ser. No. 12/736,814, filed12 Nov. 2010, entitled “Fluorescence Detection And Deactivation OfPoison Oak Oil”, and U.S. Continuation patent application Ser. No.13/714,899 filed 14 Dec. 2012, entitled “Fluorescence Detection AndDeactivation Of Poison Oak Oil”, each of which is herein incorporated byreference in its entirety for all purposes.

This invention was made partly using funds from United States NationalScience Foundation (NSF) research grant No. CHE-0453126. The FederalGovernment has certain rights to this invention.

FIELD OF THE INVENTION

The invention provides compositions, kits, and methods of using thecompositions and kits for detecting, deactivating, degrading,immunogenic compounds from poison oak and poison ivy.

BACKGROUND

Urushiol-induced allergic contact dermatitis in the United States mostcommonly results from unexpected exposure to oils from plants in thesumac Family Anacardiaceae. Approximately 10 to 50 million Americanssuffer from rashes resulting from exposure every year. In particular,the genus Toxicodendron species (which include Western and Easternpoison oak T. diversilobum, poison ivy T. radicans, and poison sumac ordogwood T. vernix) are distributed widely across North America. Othersources of urushiol include poison wood (in Florida and the Bahamas),and the sap (kiurushi) of the Asian lacquer tree (Toxicodendronverniciflua) used as a varnish in Japanese lacquer ware, and cashew nutshells. (See, for example, Tucker and Swan (1998) NEJM, 339(4): 235.)

Reaction to urushiol is an immunological response to the bio-oxidizedform of urushiol (the ortho-quinone). Approximately 50-70% of the U.S.population is either allergic to urushiol, or will become allergic to itupon sensitization by repeated exposure. Symptoms of allergic contactdermatitis from urushiol exposure (often referred to as Rhus dermatitis)vary from a mild annoyance to weeks of irritation and pain.Occasionally, exposure can lead to nephropathy and even to fatalsystemic anaphylaxis. The monetary cost due to worker disability fromurushiol-induced injuries is substantive: in the states of California,Washington and Oregon, it has been estimated that up to one third offorestry workers are temporarily disabled by poison oak dermatitis eachyear. In California, the medical costs associated with poison oakinjuries accounts for up to 1% of the annual workers' compensationbudget. It has been estimated that Toxicodendron dermatitis isresponsible for 10% of the total U.S. Forest Services lost-timeinjuries. In 1988, NIOSH estimated that 1.07-1.65 million occupationalskin injuries occurred yearly, with an estimated annual rate of 1.4 to2.2 cases per 100 workers (8) the costs attributable to lostproductivity, medical payments, and disability payments are very high.(See U.S. Centers for Disease Control; Leading work-related diseases andinjuries—United States. MMWR, 1986 335:561-563).

Chemically, urushiol is a name given to a collection of relatedcompounds that are 3-substutitued catechols (1,2-benenediols), in whichthe long hydrophobic chain is a linear C₁₅ or C₁₇ alkyl chain containing0-4 degrees of cis unsaturation (FIG. 1). The catechols with two, three,and four carbon-carbon double bonds (2-4 degrees of unsaturation) seemto be the most virulent in eliciting an allergic response. Each of thedifferent members of the Toxicodendron species contain mixtures of theC₁₅ or C₁₇ alkyl chains, with various degrees of unsaturation.

They all share the catechol functionality in common, and a long, greasyalkyl chain that facilitates migration into the skin. In addition todirect contact with the toxic plants, exposure commonly occurs bytransfer from animal fur, contaminated clothing, garden tools,fire-fighting equipment, forestry and sports equipment. There are a fewcommercially available products that can be applied prophylactically toprotect the skin by creating a physical barrier using organoclays (forexample, a lotion containing quaternium-18 bentonite is commerciallyavailable as IVYBLOCK from Enviroderm Pharmaceuticals, Inc.). However,the success of this strategy requires advanced planning. By far themajority of allergic contact dermatitis cases from urushiol result fromunexpected exposure.

A number of methods to treat poison ivy or poison oak have beeninvestigated, including hyposensitization, but this process is involvedand can have unfavorable side effects. Studies towards an immunologicalapproach to desensitization have been pursued, but have not yet reacheda level of practical application. The best treatment to date is to avoidcontact with urushiol. As most patients are unaware that they have hadcontact with urushiol, a low cost, quick and inexpensive method ofdetection is warranted. There are many recommended methods to removeurushiol after recent contact, including water, soapy water, organicsolvents, and a variety of commercially available solubilizing mixturesincluding TECHNU, IVYCLEANSE, ALL-STOP, ZANFEL (comprising fatty acid,alcohol, and the surfactant sodium lauroyl sarcosinate), and even DIALultra dishwashing soap. Thus the ability to detect urushiol before ittransverses the skin will be extremely valuable in mitigating thesuffering caused by contact with the various Toxicodendron species. Inaddition, continued re-exposure (chronic exposure) from repeatedintroduction of the oil to the patient (from door handles, shoelaces,etc.) is a considerable problem. As little as 0.001 mg of urushiol isenough to cause allergic contact dermatitis.

Treatment of the contact dermatitis usually involves a course of topicaland/or enteric treatments with hydrocortisones, β-methasone, and othersimilar corticosteroids. Repeated exposure to either the originalallergen or to a similar allergen can result in a severe hypersensitiveimmunoreaction, that is often extremely painful and, occasionally,fatal. There is therefore a particular need in the art for compounds andmethods of treatment that can remove the allergen(s) prior to inductionof an immune and/or allergic response, that can prevent the binding ofthe allergen(s) to an immunoglobulin or a cell-surface receptor, and/orthat can be used to rapidly detect the presence of such allergen(s) sothat other precautions may be used to remove the allergen(s) from thearea of contact.

There is therefore a need in the art to provide for compositions andmethods for detecting the presence of urushiol, inactivating urushiol,and removing urushiol from substrates (including, for example, skin andclothing).

BRIEF DESCRIPTION OF THE INVENTION

The invention is drawn to novel methods, kits, sprays (including aerosolsparays) and compositions for detecting active compounds present in oilsthat are found in poison oak, poison ivy, poison sumac, cashew nut, andrelated plants. The methods disclosed herein may also be used to detectother catechols, both synthetic and those found in nature. The inventionalso is drawn to compositions that may be used to detect said activecompounds using fluorescence. In one embodiment the methods of theinvention may be used to detect catechols and alkyl-substitutedcatechols, such as, for example, urushiol, catechin, epicatechin,gallocatechin, epigallocatechin, epigallocatechin-3-gallate, and thelike; and chatecholamines, such as, for example, epinephrine,norepinephrine, dopamine, dihydroxyphenylalanine (DOPA), and the like.

The invention provides methods for detecting, treating, and deactivatingthe antigenic and/or allergenic compounds that induce urushiol-inducedcontact dermatitis. In one embodiment the method may be used fortreating, deactivating, and/or detecting alk(en)yl catechols, and/oralk(en)yl resorcinols.

The invention may be used by clinicians, nursing staff, paramedics,emergency rescue team members, the military, firefighters, forestrypersonnel, lumberworkers, hunters, mountaineers, hikers, anglers, andthe like. In one embodiment, the invention is a kit comprising theelements disclosed herein and a set of instructions of how to use thekit, wherein the kit is used for detecting, treating, and/ordeactivating a catechol. The kit can be used, for example, in the home,in the field, in a camp, in a clinic, in a hospital, in an emergencyroom, and the like.

The invention provides a kit for detecting a catechol, the kitcomprising a vessel, the vessel shaped and adapted for confining acomposition, the composition further comprising a boron composition, afirst nitroxide, and a second nitoxide, and an applicator. In oneembodiment the boron composition comprises a hydrophobic alkyl group. Inanother embodiment the second nitroxide is a profluorescent nitroxide.In a preferred embodiment the applicator is a spray applicator. In amost preferred embodiment the catechol is urushiol. In one alternativeembodiment, the kit can also comprise an aerosol propellant. In anotherembodiment the kit comprises a lamp.

In a preferred embodiment, the invention provides a method for detectinga catechol in a sample, the method comprising the steps of (i)contacting a boron composition and a nitroxide with the sample (ii)allowing the boron composition to react with the catechol in the samplethereby creating a catecholborane; (iii) allowing a first nitroxide toreact with the catecholborane thereby generating an alkyl radical and anitroxide-catecholborane complex; (iv) allowing the alkyl radical toreact with a second nitroxide thereby creating an alkoxyamine; (v)measuring the amount of alkoxyamine, nitroxide-catecholborane complex,or an alkoxyamine hydrolysis product so created; the method resulting indetecting the catechol in the sample. In one embodiment the boroncomposition comprises a hydrophobic alkyl group. In a preferredembodiment, the catecholborane is a B-alkyl catecholborane. In anotherpreferred embodiment the alkyl group is selected from the groupconsisting of a hydrophobic alkyl group and a hydrophilic alkyl group.In a yet alternative embodiment the nitroxide is a profluorescentnitroxide. More preferably, the nitroxide is tetramethylpiperidinyloxy(TEMPO). In a more preferred embodiment the profluorescent nitroxide isdansyl amino-TEMPO. In another preferred embodiment the sample isselected from the group consisting of an area of a subject's skin,clothing, boots, pets, camping gear, tools, and other outdoor equipment.In another preferred embodiment the sample is selected from the groupconsisting of a plant tissue, a plant extract, a plant tissue extract,an animal tissue, an animal extract, an animal tissue extract, and ananimal fluid. In a more preferred embodiment the plant tissue is from aplant selected from the group consisting of poison oak, poison ivy,poison sumac, mango, cashew nut, and lac tree.

The invention further provides the methods as disclosed herein whereinthe nitroxide further comprises a fluorescent compound, the fluorescentcompound selected from the group consisting of a hydrophobic fluorescentorganic molecule, a hydrophilic fluorescent organic molecule, and afluorescent quantum-dot nanoparticle.

In one embodiment the method comprises the measuring the amount ofalkoxyamine so created using a photon source that results influorescence of the alkoxyamine and the nitroxide-catecholboranecomplex, wherein the fluorescence is visible to the naked eye. In apreferred embodiment the measuring of the amount of alkoxyamine socreated is performed using a photon source that induces fluorescence ofthe alkoxyamine and the nitroxide-catecholborane complex, wherein thefluorescence is detected by a photometer. In a more preferred embodimentthe fluorescence comprises photons having a wavelength of between about250 and 600 nm. In one embodiment the photon source is a lamp. In apreferred embodiment the lamp is a hand-held lamp. In an alternativeembodiment the photon source is the sun. The method may also furthercomprise measuring hydroxylamine complexed with boron or freehydroxylamine created by hydrolysis.

In a preferred embodiment of the invention the catechol is selected fromthe group consisting of urushiol, catechin, epicatechin, gallocatechin,epigallocatechin, epigallocatechin-3-gallate, and catecholaminesepinephrine, norepinephrine, dopamine, and dihydroxyphenylalanine(DOPA). In a more preferred embodiment the catechol is urushiol.

The method may further comprise the step of reacting the alkyl radicalwith a profluorescent nitroxide having a fluorescent tag, wherein thefluorescent tag is selected from the group consisting of an organicfluorophore and Cd—Se nanoparticle. In another embodiment the method mayfurther comprise the step of measuring the amount of thenitroxide-catecholborane complex. In another embodiment the methodfurther comprises the step of measuring the amount of hydroxylaminehydrolysis product. In a yet other embodiment the method furthercomprises the step of measuring the amount of alkoxyamine product.

The invention also provides for a method for deactivating a catechol ina sample, the method comprising the steps of (i) contacting a boroncomposition and an oxygen-containing molecule with the sample (ii)allowing the boron composition to react with the catechol in the samplethereby creating a catecholborane; the method resulting in deactivatingthe catechol in the sample. In one embodiment the boron compositioncomprises a hydrophobic alkyl group. In a preferred embodiment, thecatecholborane is a B-alkyl catecholborane. In another preferredembodiment the alkyl group is selected from the group consisting of ahydrophobic alkyl group and a hydrophilic alkyl group. In a yetalternative embodiment the nitroxide is a profluorescent nitroxide. Morepreferably, the nitroxide is tetramethylpiperidinyloxy (TEMPO). In amore preferred embodiment the profluorescent nitroxide is dansylamino-TEMPO. In another preferred embodiment the sample is selected fromthe group consisting of an area of a subject's skin, clothing, boots,pets, camping gear, tools, and other outdoor equipment. In anotherpreferred embodiment the sample is selected from the group consisting ofa plant tissue, a plant extract, a plant tissue extract, an animaltissue, an animal extract, an animal tissue extract, and an animalfluid. In a more preferred embodiment the plant tissue is from a plantselected from the group consisting of poison oak, poison ivy, poisonsumac, mango, cashew nut, and lac tree.

In one preferred embodiment the oxygen-containing molecule comprises anitroxide. The invention further provides the methods as disclosedherein wherein the nitroxide further optionally comprises a fluorescentcompound, the fluorescent compound selected from the group consisting ofa hydrophobic fluorescent organic molecule, a hydrophilic fluorescentorganic molecule, and a fluorescent quantum-dot nanoparticle.

In one embodiment the method comprises the measuring the amount ofalkoxyamine so created using a photon source that results influorescence of the alkoxyamine and the nitroxide-catecholboranecomplex, wherein the fluorescence is visible to the naked eye. In apreferred embodiment the measuring of the amount of alkoxyamine socreated is performed using a photon source that induces fluorescence ofthe alkoxyamine and the nitroxide-catecholborane complex, wherein thefluorescence is detected by a photometer. In a more preferred embodimentthe fluorescence comprises photons having a wavelength of between about250 and 600 nm.

The method may also further comprise measuring hydroxylamine complexedwith boron or free hydroxylamine created by hydrolysis.

In a preferred embodiment of the invention the catechol is selected fromthe group consisting of urushiol, catechin, epicatechin, gallocatechin,epigallocatechin, epigallocatechin-3-gallate, and catecholaminesepinephrine, norepinephrine, dopamine, and dihydroxyphenylalanine(DOPA). In a more preferred embodiment the catechol is urushiol.

The invention also provides for a boron composition, the boroncomposition comprising a reactive moiety that reacts with a catecholwith a rate constant, k, of at least 0.2 M⁻¹s⁻¹ and wherein the reactionproduces a stable chatecholborane.

The invention provides for a pharmaceutical composition, thepharmaceutical composition comprising a boron composition, wherein theboron composition comprises a hydrophobic alkyl group. In one embodimentthe alkyl group is selected from the group consisting of a hydrophobicalkyl group and a hydrophilic alkyl group. In another embodiment thepharmaceutical composition comprises a boron composition in an effectiveamount for the treatment of poison oak oil-induced contact dermatitis.In a preferred embodiment the poison oak oil comprises a catechol. In amore preferred embodiment the catechol is urushiol.

The invention provide a topical composition, the topical compositioncomprising an effective amount of a boron composition and a suitableexcipient, carrier, or combination thereof, the boron compositioncomprising an alkylboronic acid having the general formula R—B(OH)₂. Inone alternative embodiment the boron composition optionally comprises atleast one B-alkyl boronic acid derivative. In another embodiment thetopical composition optionally containing xanthan gum or gellan gum. Ina more preferred embodiment the boron composition is present in anamount selected from the group consisting of from about 99.5% to about0.001%, from about 95% to about 0.1%, and from about 90% to about 0.5%,by weight, based on the total combined weight of the boron compositionthereof, not including other excipient, carrier, or combination thereof.In a most preferred embodiment the topical composition comprises a boroncomposition in an effective amount for the detection of a catechol inpoison oak oil.

The invention further provides a topical medicament, the topicalmedicament comprising a boron composition, the boron compositioncomprising an alkylboronic acid having the general formula R—B(OH)₂, anitroxide, and a suitable excipient, carrier, or combination thereof,and where R is selected from the group consisting of a hydrophobic alkylgroup and a hydrophilic alkyl group. In an alternative embodiment theboron composition optionally comprises at least one B-alkyl boronic acidderivative. In a more preferred embodiment the nitroxide is aprofluorescent nitroxide. In a more preferred embodiment the topicalmedicament comprises a boron composition in an effective amount for thedetection of a catechol in poison oak oil to avoid induced contactdermatitis. In another more preferred embodiment the topical medicamentcomprises a boron composition in an effective amount for the treatmentof poison oak oil-induced contact dermatitis.

In one embodiment, the invention provides a method for detecting,treating, and deactivating alk(en)yl catechols, and/or alk(en)ylresorcinols using a boron compound bearing a hydrophobic alkyl group andan at least one equivalent of profluorescent nitroxide are that aremixed in solution or on a substrate. In one preferred embodiment, theprofluorescent nitroxide is a nitroxide with a short tether to afluorescent dye, wherein the dye is quenched in the presence of the freenitroxide. In an alternative embodiment the boron compound furthercomprises an alkyl boronic acid or alkyl boronic acid derivative. Inanother alternative embodiment the boron compound further comprises atleast one leaving group. In yet another alternative embodiment, theboron compound further comprises two leaving groups.

In one embodiment the invention provides a method for detecting,treating, and deactivating alk(en)yl catechols, and/or alk(en)ylresorcinols, wherein the method results in producing a fluorescentcompound that fluoresces when illuminated and wherein the fluorescenceis induced by photons having a wavelength of between about 250 and 600nm. In one embodiment the fluorescence can be, for example, between 250and 300 nm, between 300 and 350 nm, between 350 and 400 nm, between 450and 500 nm, between 500 and 550 nm, and between 550 and 600 nm. In thealternative, the method results in producing a fluorescent compound thatfluoresces when illuminated with light in the visible spectrum andwherein the fluorescence is induced by photons having a wavelength ofbetween about 600 and 750 nm. In one embodiment the fluorescence can be,for example, between 600 and 650 nm, between 650 and 700 nm, and between700 and 750 nm.

In another alternative embodiment, the nitroxide can comprise afluorescent tag such as, for example, a fluorescent organic compound,such as dansyl, 3-hydroxy-2-methyl-4-quinolinecarboxylic ester, acoumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, anoxazine, bimane, 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonicacid (SITS) and related stilbene derivatives, and the isothiocyanate ofpyrenetrisulfonic acid, fluorescein, acryoldan, rhodamine,dipyrrometheneboron difluoride (BODIPY), acridine orange, eosin,acridine orange,1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridiniumbromide (PyMPO), alexa fluor 488, alexa fluor 532, alexa fluor 546,alexa fluor 568, alexa fluor 594, alexa fluor 555, alexa fluor 633,alexa fluor 647, alexa fluor 660 and alexa fluor 680, or the like, or aquantum-dot nanoparticle. In the present invention, a non-limited listof quantum dot nanoparticles includes cadmium sulfide (CdS), cadmiumselenide (CdSe), zinc sulfide (ZnS), zinc oxide (ZnO), lead sulfide(PbS), zinc selenide (ZnSe), GaAS, and InP. (Lakowicz et al., Anal.Biochem., 2000, 280: 128-136.

The invention further provides use of a composition comprising a boroncomposition for the manufacture of a composition for detecting acatechol. In one embodiment the boron composition comprises analkylboronic acid having the general formula R—B(OH)₂, a nitroxide, anda suitable excipient, carrier, or combination thereof, and where R isselected from the group consisting of a hydrophobic alkyl group and ahydrophilic alkyl group. In one alternative embodiment the boroncomposition optionally comprises at least one B-alkyl boronic acidderivative. In a preferred embodiment the nitroxide is a profluorescentnitroxide. In another preferred embodiment the composition comprises aboron composition in an effective amount for the detection of a catecholin poison oak oil.

The invention can be used in a variety of embodiments, for example, foruse as chemical sensors and molecular specific deactivating agents. Theinvention can be used in phototherapy for treatment of an inflammatoryresponse and other disorders. The invention can also be used as a sensorthat detects molecules. The invention is of particular use in the fieldsof clinical diagnosis, clinical therapy, clinical treatment, andclinical evaluation of various diseases and disorders, in the field ofconsumer goods, for example, over-the-counter medications, balms,ointments, etc., and diagnostic kits, manufacture of compositions foruse in the treatment of various diseases and disorders, for use inmolecular biology, structural biology, cell biology, molecular switches,molecular circuits, and molecular computational devices, and themanufacture thereof.

In one embodiment, the composition comprises a surface stabilizer. Inanother alternative embodiment the composition comprises at least twosurface stabilizers. In a preferred embodiment, the surface stabilizeris selected from the group consisting of an anionic surface stabilizer,a cationic surface stabilizer, a zwitterionic surface stabilizer, and anionic surface stabilizer.

In another preferred embodiment, the surface stabilizer is selected fromthe group consisting of cetyl pyridinium chloride, gelatin, casein,phosphatides, dextran, glycerol, gum acacia, cholesterol, tragacanth,stearic acid, benzalkonium chloride, calcium stearate, glycerolmonostearate, cetostearyl alcohol, cetomacrogol emulsifying wax,sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castoroil derivatives, polyoxyethylene sorbitan fatty acid esters,polyethylene glycols, dodecyl trimethyl ammonium bromide,polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodiumdodecylsulfate, carboxymethylcellulose calcium, hydroxypropylcelluloses, hypromellose, carboxymethylcellulose sodium,methylcellulose, hydroxyethylcellulose, hypromellose phthalate,noncrystalline cellulose, magnesium aluminum silicate, triethanolamine,polyvinyl alcohol, polyvinylpyrrolidone,4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde, poloxamines, a charged phospholipid,dioctylsulfosuccinate, dialkylesters of sodium sulfosuccinic acid,sodium lauryl sulfate, alkyl aryl polyether sulfonates, mixtures ofsucrose stearate and sucrose distearate,p-isononylphenoxypoly-(glycidol), decanoyl-N-methylglucamide; n-decylβ-D-glucopyranoside; n-decyl β-D-maltopyranoside; n-dodecylβ-D-glucopyranoside; n-dodecyl β-D-maltoside;heptanoyl-N-methylglucamide; n-heptyl-β-D-glucopyranoside;n-heptylβ-D-thioglucoside; n-hexyl β-D-glucopyranoside;nonanoyl-N-methylglucamide; n-noyl β-D-glucopyranoside;octanoyl-N-methylglucamide; n-octyl-β-D-glucopyranoside; octylβ-D-thioglucopyranoside; lysozyme, PEG-phospholipid, PEG-cholesterol,PEG-cholesterol derivative, and PEG-vitamin A.

In another alternative embodiment, the cationic surface stabilizer isselected from the group consisting of a polymer, a biopolymer, apolysaccharide, a cellulosic, an alginate, a nonpolymeric compound, anda phospholipid.

In another alternative embodiment, the surface stabilizer is selectedfrom the group consisting of cationic lipids, polymethylmethacrylatetrimethylammonium bromide, sulfonium compounds,polyvinylpyrrolidone-2-dimethylaminoethyl methacrylate dimethyl sulfate,hexadecyltrimethyl ammonium bromide, phosphonium compounds, quarternaryammonium compounds, benzyl-di(2-chloroethyl)ethylammonium bromide,coconut trimethyl ammonium chloride, coconut trimethyl ammonium bromide,coconut methyl dihydroxyethyl ammonium chloride, coconut methyldihydroxyethyl ammonium bromide, decyl triethyl ammonium chloride, decyldimethyl hydroxyethyl ammonium chloride, decyl dimethyl hydroxyethylammonium chloride bromide, C₁₂₋₁₅ dimethyl hydroxyethyl ammoniumchloride, C₁₂₋₁₅-dimethyl hydroxyethyl ammonium chloride bromide,coconut dimethyl hydroxyethyl ammonium chloride, coconut dimethylhydroxyethyl ammonium bromide, myristyl trimethyl ammonium methylsulphate, lauryl dimethyl benzyl ammonium chloride, lauryl dimethylbenzyl ammonium bromide, lauryl dimethyl (ethenoxy)4 ammonium chloride,lauryl dimethyl (ethenoxy)₄ ammonium bromide, N-alkyl(C₁₂₋₁₈)dimethylbenzyl ammonium chloride, N-alkyl(C₁₄₋₁₈)dimethyl-benzyl ammonium chloride, N-tetradecylidmethylbenzy-1ammonium chloride monohydrate, dimethyl didecyl ammonium chloride,N-alkyl and (C₁₂₋₁₄) dimethyl 1-napthylmethyl ammonium chloride,trimethylammonium halide, alkyl-trimethylammonium salts,dialkyl-dimethylammonium salts, lauryl trimethyl ammonium chloride,ethoxylated alkyamidoalkyldialkylammonium salt, an ethoxylated trialkylammonium salt, dialkylbenzene dialkylammonium chloride,N-didecyldimethyl ammonium chloride, N-tetradecyldimethylbenzylammonium, chloride monohydrate, N-alkyl(C₁₂₋₁₄) dimethyl1-naphthylmethyl ammonium chloride, dodecyldimethylbenzyl ammoniumchloride, dialkyl benzenealkyl ammoniumchloride, lauryl trimethylammonium chloride, alkylbenzyl methyl ammonium chloride, alkyl benzyldimethyl ammonium bromide, C₁₂ trimethyl ammonium bromides, C₁₅trimethyl ammonium bromides, C₁₇ trimethyl ammonium bromides,dodecylbenzyl triethyl ammonium chloride, poly-diallyldimethylammoniumchloride (DADMAC), dimethyl ammonium chlorides, alkyldimethylammoniumhalogenides, tricetyl methyl ammonium chloride, decyltrimethylammoniumbromide, dodecyltriethylammonium bromide, tetradecyltrimethylammoniumbromide, methyl trioctylammonium chloride, polyquaternium 10,tetrabutylammonium bromide, benzyl trimethylammonium bromide, cholineesters, benzalkonium chloride, stearalkonium chloride compounds, cetylpyridinium bromide, cetyl pyridinium chloride, halide salts ofquaternized polyoxyethylalkylamines, quaternized ammonium salt polymers,alkyl pyridinium salts; amines, amine salts, amine oxides, imideazolinium salts, protonated quaternary acrylamides, methylatedquaternary polymers, and cationic guar.

The invention also provides for a chemical spray that can be used in thefield to allow the detection of urushiol in conjunction with the use ofa fluorescent lamp. In one embodiment the amount of urushiol detected isin the range of between about 0.1-100 μg. In a preferred embodiment, theamount of urushiol detected is in the range of between about 1-10 μg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the chemical formulae of chatechol and exemplaryurushiols.

FIG. 2 illustrates how B-alkyl catechol borane species react with oxygenradicals to expel alkyl radicals (adapted from Darency and Renaud, 2006,Top. Curr. Chem., 263: 71-106; Cadot et al. 2002, JOC, 67: 7193-7202;Baban et al. 1986, J. Chem. Soc., Perkin Trans. 2: 157).

FIG. 3 illustrates a modified Brown and Negishi reaction that maycomprise chain transfers with PTOC-OMe for radical acceptors (Brown andNegishi, 1971, J. Am. Chem. Soc. 93: 3777; Suzuki et al. 1969, J. Chem.Soc., Chem. Commun., 17: 1009; Forster 1999, PhD Thesis, University deFribourg, Switzerland, Diss Nr. 1242; Ollivier and Renaud 1999, Chem.Eur. J., 5: 1468; Kumli and Renaud, 2006, Org. Lett. 8: 5861; Olivierand Renaud, 2000, Angew. Chem. Int. Ed. 39: 925).

FIG. 4 illustrates novel methods for detecting poison oak oil (includingpoison ivy, sumac oil, and lac tree extracts) that are present upon asubstrate by chemical generation of fluorescence.

FIG. 5 illustrates an exemplary reaction between a nitroxide (forexample, TEMPO) and a catechol that results in anitroxide-catecholborane.

FIG. 6 illustrates an exemplary reaction between a profluorescentnitroxide and a catechol that results in a fluorescentnitroxide-catecholborane.

FIG. 7A illustrates details of another exemplary reaction between eithera borane compound (top), a catecholborane (middle and bottom), and anitroxide or profluorescent nitroxide that results in no reaction (top),production of a nitroxide-catecholborane (middle), or production of afluorescent nitroxide-catecholborane (bottom). FIG. 7B illustratesdetails of how a reaction between a profluorescent nitroxide in thepresence of phenylhydrazine results in production of a fluorescentcompound.

FIG. 8 illustrates that addition of an oxygen radical to analkylcatecholborane forms a perboryl radical 5, visible by ESR

FIG. 9 illustrates that addition of nitroxide to an alkylcatecholboraneforms a perboryl radical 6, which fragments to generate an alkylradical. A second equivalent of nitroxide reacts with the alkyl radicalto form alkoxyamine 8.

FIG. 10 shows exemplary profluorescent nitroxides: the free nitroxidequenches fluorescence of a closely tethered fluorophore; fluorescence isrestored upon reaction to from the alkoxyamine or hydroxylamine.

FIG. 11 illustrates a reaction sequence that may detect catechol usingprofluorescent nitroxide addition to alkylcatecholborane 13.

FIG. 12 illustrates use of profluorescent Dansyl amino-TEMPO:preparation, reduction, and formation of radical trapping product 17.

FIG. 13 shows an exemplary reaction of a model alkyl-catecholborane 19with two equivalents of nitroxide: both alkoxyamine 20 and hydroxylamine21 were isolated from the reaction mixture.

FIG. 14 illustrates reaction of profluorescent Dansyl amino-TEMPO 16with n-butylcatecholborane 19 in toluene to give fluorescentn-butylalkoxyamine 22 (A): paper towel spot test shows fluorescence ofalkoxyamine 22 (B).

FIG. 15 shows the in situ formation of n-butylcatecholborane 19 andsubsequent reaction to form fluorescent 22 (A) in one pot (B).

FIG. 16 illustrates common classes of readily synthesized stablenitroxides.

FIG. 17 illustrates a general synthesis pioneered by Hideg and Keana forthe preparation of proxyl nitroxides 42.

FIG. 18 shows the synthesis of the pyrene proxyl profluorescentnitroxide 44.

FIG. 19 illustrates a few representative known profluorescentnitroxides.

FIG. 20 illustrates the excitation and emission spectra ofprofluorescent nitroxide 12 and fluorescent N-alkoxyamine 28 in DMSO.

FIG. 21 shows a hydroboration route to prepare n-alkylboronic acids 25

FIG. 22 illustrates representative pyrogallols and catechols commonlyfound in foods such as red wine, tea, and chocolate: note that compounds48 and 49 are polyols, and are thus aqueous rather than organic soluble.

FIG. 23 illustrates a slow reduction of nitroxide by catechol; rapidreoxidation of the hydroxylamine to the nitroxide with PbO₂.

FIG. 24 illustrates fluorescence quenching and recovery upon addition ofcatechol to profluorescent nitroxide 12, with and without addition ofPbO₂ as a reoxidant.

FIG. 25 illustrates how exemplary mild oxidants can rapidly oxidizehydroxylamine to nitroxide but that do not oxidize catechol to quinone.

FIG. 26 illustrates detection of urushiol on leaves of poison oak. A:Fresh Poison Oak triad of leaves; B: Print of the same leaves on a papertowel after treatment with Fl-NitO., nBuB(OH)₂ and catalytic PbO₂ inacetone.

DETAILED DESCRIPTION OF THE INVENTION

In order to develop a system to selectively detect catechols in thepresence of other alcohols and diols (such as sugars), a reaction thattakes place with catechols but not with other alcohols was required. Inthe field of organic free radical chemistry, alkylcatecholboranes havebeen used to selectively generate alkyl radicals upon reaction withoxygen radicals. The efficacy of this oxygen radical additionspecifically to alkylcatecholboranes is due to de-localization of theunpaired electron of the perboryl species 5 into the aromatic ring (FIG.8). Direct ESR evidence for this delocalized perboryl radical 5 below270 K was observed by Roberts (Baban et al., J. Chem. Soc. PerkinTransact. 1986, 2(1): 157-161). A number of very useful syntheticmethodologies have been developed from this chemistry. Key to thisproposal is the work by Renaud, in which addition of two equivalents ofthe oxygen radical TEMPO 7, a commercially available persistentnitroxide radical, results in formation of the carbon radical trappingproduct, alkoxyamine 8 (FIG. 9).

In order to design a visual indicator of the reaction of nitroxides withalkylcatecholboranes, profluorescent nitroxides are used. Profluorescentnitroxides 10 (sometimes referred to as “pre-fluorescent nitroxides”)are nitroxides bearing a short covalent tether to a fluorophore. Thefree nitroxide quenches the fluorescence. Upon reaction of the nitroxidemoiety to form an alkoxyamine 11 or a hydroxylamine (or any othernon-nitroxide product), the fluorescence is no longer quenched,restoring fluorescence to the product (FIG. 10). Profluorescentnitroxides have been utilized as sensors of nitric oxide, antioxidants,reactive oxygen species, carbon radicals, cationic metals, viscosityprobes, as a chemical logic gate, and in the development ofphotomagnetic materials. (See Ivan, M. G.; Scaiano, J. C.,Photochemistry and Photobiology 2003, 78, (4), 416-419; Hornig, F. S.;Korth, H. G.; Rauen, U.; de Groot, H.; Sustmann, R., Helvetica ChimicaActa 2006, 89, (10), 2281-2296; Lozinsky, E. M.; Martina, L. V.; Shames,A. I.; Uzlaner, N.; Masarwa, A.; Likhtenshtein, G. I.; Meyerstein, D.;Martin, V. V.; Priel, Z., Analytical Biochemistry 2004, 326, (2),139-145; Meineke, P.; Rauen, U.; de Groot, H.; Korth, H. G.; Sustmann,R., Chemistry—a European Journal 1999, 5, (6), 1738-1747; Meineke, P.;Rauen, U.; de Groot, H.; Korth, H. G.; Sustmann, R., BiologicalChemistry 2000, 381, (7), 575-582; Blough, N. V.; Simpson, D. J.,Journal of the American Chemical Society 1988, 110, (6), 1915-1917;Lozinsky, E.; Martin, V. V.; Berezina, T. A.; Shames, A. I.; Weis, A.L.; Likhtenshtein, G. I., Journal of Biochemical and Biophysical Methods1999, 38, (1), 29-42; Tang, Y. L.; He, F.; Yu, M. H.; Wang, S.; Li, Y.L.; Zhu, D. B., Chemistry of Materials 2006, 18, (16), 3605-3610; Hideg,E.; Kalai, T.; Kos, P. B.; Asada, K.; Hideg, K., Photochemistry andPhotobiology 2006, 82, (5), 1211-1218; Aspee, A.; Garcia, O.; Maretti,L.; Sastre, R.; Scaiano, J. C., Free radical reactions in poly(methylmethacrylate) films monitored using a prefluorescent quinoline-TEMPOsensor. Macromolecules 2003, 36, (10), 3550-3556; Aspee, A.; Maretti,L.; Scaiano, J. C., Photochemical & Photobiological Sciences 2003, 2,(11), 1125-1129; Ballesteros, O. G.; Maretti, L.; Sastre, R.; Scaiano,J. C., Macromolecules 2001, 34, (18), 6184-6187; Blinco, J. P.;McMurtrie, J. C.; Bottle, S. E., European Journal of Organic Chemistry2007, 4638-4641; Coenjarts, C.; Garcia, O.; Llauger, L.; Palfreyman, J.;Vinette, A. L.; Scaiano, J. C., Journal of the American Chemical Society2003, 125, (3), 620-621; Dang, Y. M.; Guo, X. Q., Applied Spectroscopy2006, 60, (2), 203-207; Fairfull-Smith, K. E.; Blinco, J. P.; Keddie, D.J.; George, G. A.; Bottle, S. E., Macromolecules 2008, 41, 1577-1580;Gerlock, J. L.; Zacmanidis, P. J.; Bauer, D. R.; Simpson, D. J.; Blough,N. V.; Salmeen, I. T., Free Radical Research Communications 1990, 10,(1-2), 119-121; Johnson, C. G.; Caron, S.; Blough, N. V., AnalyticalChemistry 1996, 68, (5), 867-872; Maurel, V.; Laferriere, M.; Billone,P.; Godin, R.; Scaiano, J. C., Journal of Physical Chemistry B 2006,110, (33), 16353-16358; Micallef, A. S.; Blinco, J. P.; George, G. A.;Reid, D. A.; Rizzardo, E.; Thang, S. H.; Bottle, S. E., PolymerDegradation and Stability 2005, 89, (3), 427-435; Nagy, V. Y.; Bystryak,I. M.; Kotelnikov, A. I.; Likhtenshtein, G. I.; Petrukhin, O. M.;Zolotov, Y. A.; Volodarskii, L. B., Analyst 1990, 115, (6), 839-841;Arye, P. P.-B.; Strashnikova, N.; Likhtenshtein, G. I., Journal ofBiochemical and Biophysical Methods 2002, 51, (1), 1-15; and Wang, H.M.; Zhang, D. Q.; Guo, X. F.; Zhu, L. Y.; Shuai, Z. G.; Zhu, D. B.,Chemical Communications 2004, (6), 670-671.)

The use of a profluorescent nitroxide with an alkylboronic acidderivative 12 is envisioned to react with catechols (such as, but notlimited to, for example, urushiol) to form alkylboronate 13: nitroxideaddition, radical 14 generation, and nitroxide trapping will generatethe fluorescent signal of alkoxyamine 15. Other alkylboronic acidderivatives will be apparent to those of skill in the art.

Catechols are a group of compounds well-known to those of skill in theart having diverse biological activities, whilst at the same time beingstructurally conservative. The invention contemplates that thecompositions and methods disclosed herein may be used to detect,inactivate, or bind to any biologically-active catechol composition. Inparticular the invention contemplates a catechol selected from the groupconsisting of urushiol, catechin, epicatechin, gallocatechin,epigallocatechin, epigallocatechin-3-gallate, and catecholaminesepinephrine, norepinephrine, dopamine, and dihydroxyphenylalanine(DOPA). One of skill in the art would consider that the structures ofcatechols are sufficiently similar that they are a well-known chemicalclass of compounds.

Profluorescent nitroxide is sometimes referred to as a pre-fluorescentnitroxide. In the presence of a catechol such as urushiol and anB-alkylboronic acid derivative, a B-alkyl catecholborionate is formed.Addition of the nitroxide to the catecholborane results in expulsion ofan alkyl radical, which is trapped by a second nitroxide, forming twofluorescent species: an alkoxyamine with a fluorescent tag, andfluorescently tagged nitroxide-catecholborane complex. In addition, thenitroxide-catecholborane may degrade to hydroxylamine that is also afluorescent compound. Use of a hand-held fluorescent lamp showsfluorescence when a catechol such as urushiol is present. This can beused as a method to detect the presence of urushiol. As a treatment,binding of the urushiol into a catecholborane complex will preventtransfer through the skin, preventing oxidation of the catechol andelicitation of an immune response, thus preventing contact dermatitis.For detecting aqueous soluble catechols such as dopamine, epinephrine,and norepinephrine, a water-soluble alkyl group is preferred on theinitial boron compound rather than a hydrophobic alkyl group.

Examples of profluorescent nitroxides may be found in the followingnon-exhaustive list of publications: Blough, 1988, JACS, 110: 1915;Bottle, 2005, Polym. Degrad. & Stability, 89: 427-435; Sciano, 2001,Macromol. 34: 6184; Ibid., 2003, JACS, 125: 620; Ibid., 2003, Photochem.Photobiol. 78: 416; Turro, 2001, Macromol., 34: 8187; Koth, 2000,Biological Chem., 381(7): 575-582; Ibid., 1999, Chem. Eur. J. 5(6):1738-1747; Ibid., 1997, Ang. IEE, 36: 1501-1503; Ibid., 2006, Helv. ChimActa, 89: 2281-2296; Hideg 2006, Photochem. Photobiol. 82: 1211; Want,2006, Chem. Mater., 18: 3605; and Dang and Guo, 2006, Appl. Spectrosc.60: 203-207,

In the present invention, a non-limited list of quantum dotnanoparticles includes cadmium sulfide (CdS), cadmium selenide (CdSe),zinc sulfide (ZnS), zinc oxide (ZnO), lead sulfide (PbS), zinc selenide(ZnSe), GaAS, and InP. (Lakowicz et al. Analytical Biochemistry, 2000,280: 128-136). Alternative suitable donor fluorophores will be apparentto those of ordinary skill without undue experimentation. For example,nitroxides tethered to such a quantum dot will quench any fluorescence;when the nitroxides react with a catechol boronate complex, thequenching effect is removed and fluorescence can occur under appropriateconditions.

Use of the Compositions for Detection of Urushiol

A composition prepared according to the present invention may beformulated as an aerosol spray, a topical cream, ointment, medicament,or a solution.

An aerosol containing approximately 0.005% to about 5.0% (w/w) each ofthe boron composition and nitroxide according to the present inventionis prepared by dissolving the compositions in absolute alcohol. Theresulting solution is then diluted in an organic solvent or purifiedwater, depending upon the hydrophobicity of the compound. Routineexperimentation by those having skill in the art can be used todetermine an effective amount for detecting a catechol in a sample.

There are several biologically very important catechols: thecatecholamines (including epinephrine, norepinephrine, and dopamine), inaddition to epicatechin (common in tea). All of these are water-soluble.Because boron species undergo dynamic exchange of alcohol ligands viatheir anionic “-ate” species in water, it is likely that thismethodology may be extrapolated to detect catechols in an aqueousenvironment. The key reaction sequence of nitroxide reacting withalkylcatecholborane is well established in non-polar organic solvents.Extension to aqueous conditions would provide a very powerful detectionmethod for catecholamines: success would depend on the lifetimes of thetricoordinate borane species compared to the predominate tetracoordinateboronate species. Water-soluble nitroxides and fluorophores are widelyknown; nitroxides have been used extensively as an EPR probe in biology.The detection of biologically important catecholamines (includingepinephrine, norepinephrine, and dopamine) in aqueous environments couldlead to powerful new methods in biomedicine.

Contact dermatitis from exposure of skin to urushiol causes agony andsuffering for tens of millions of Americans each year, making this animportant human health issue in North America. Urushiol can beeffectively removed from skin, clothes and equipment, but only if it isknown where this invisible contamination is located. The inventioncomprises a fluorescence detection method: a spray containing aprofluorescent nitroxide and an alkylboronate derivative in an organicsolvent will react selectively with urushiol to form a fluorescentN-alkoxyamine. An inexpensive UV light can then be used to pinpoint thepresence of urushiol, to prevent or mitigate exposure to skin.Preliminary results with catechol confirm that the key reaction works asexpected, and that a highly fluorescent signal is generated.Optimization of the profluorescent nitroxide (both the fluorophore andnitroxide structures), solvent and fine-tuning of the alkyl group on theboronic acid are undertaken. The invention provides a clear benefit tosociety, including private outdoors enthusiasts, forestry workers,emergency rescue personnel, military personnel, and others who come incontact with poison oak, poison ivy, or sumac.

The invention also may be used to deactivate a chatechol, such asurushiol, using the methods disclosed herein. In certain case theproduct, such as B-alkyl catecholboronate or alkycatecholborane, may bechemically unstable and the composition may hydrolyse to the products,chatechol and the alkylboronate derivative, for example. It iscontemplated that such hydrolysis may be impeded or decelerated in thepresence of environmental modulators, such as a hydrophobic composition,a hydrophilic composition, a buffer composition, or the like. Suchenvironmental modulators can be sugars, carbohydrates, proteins,peptides, glycopeptides, glycolipids, and glycophospholipids; organiccompositions, such as organic acids, organic salts, organic bases, orthe like, lipids, phospholipids, or fatty acids; chemical stabilizers,or the like, or any combination thereof. Such compositions may be usedto formulate a topical medicament or topical composition that is used toreduce or eliminate the effects of poison oak oil-induced contactdermatitis.

In addition, the formulation or aerosol can comprise a solvent, thesolvent comprising a polar organic solvent, a non-polar organic solvent,an aqueous solvent, or a non-aqueous solvent.

The invention will be more readily understood by reference to thefollowing examples, which are included merely for purposes ofillustration of certain aspects and embodiments of the present inventionand not as limitations.

EXAMPLES Example I Preparation and Testing of Fluororescent Compounds

We have prepared the known profluorescent nitroxide Dansyl amino-TEMPO16. As reported, the free nitroxide quenches fluorescence; the insert ofFIG. 12 shows the reaction of the nitroxide to form either thehydroxylamine 17 (vial shown) or the n-butylalkoxyamine 18 (not shown)restores the fluorescence to the naked eye upon irradiation with a longwave-length UV lamp at 366 nm (A hand-held UV lamp typically used forviewing thin layer chromatography plates was utilized in thesephotographs).

As an initial model, B-n-butylcatecholborane 19 was pre-formed usingDean Stark conditions, and then allowed to react with two equivalents ofTEMPO 7 (FIG. 13). The expected N-n-butyloxyamine 20 was formed as amixture with the hydroxylamine 21, confirming the chemistry developed byRenaud. Hydroxylamine 21 is presumably formed by hydrolysis of thenitroxide boronic ester complex.

This reaction was repeated with the profluorescent Dansyl amino-TEMPO 16(FIG. 14A). The reaction mixture was strongly fluorescent in which adrop of solution was put on a paper towel; illumination with a thinlayer chromatography (TLC) long-wavelength lamp clearly showed a strongfluorescent signal for the alkoxyamine 28 (see FIG. 14B). Similar dropsof solution containing the profluorescent nitroxide 16 and a controlmixture of the profluorescent nitroxide mixed with n-butylboronic acidgave no detectible signal. Isolation and characterization of thefluorescent n-butylalkoxyamine 22 confirmed that the reaction hadoccurred as predicted.

Example II Fluorescence Detection of Catechol

In order to form alkylcatecholborane 13 from free catechol under ambientconditions, we initially believed it would be necessary to convert thehydroxyl groups on an alkylboronic acid to better leaving groups.However, early work by Brown indicated that alkylboronic acids reactreversibly with catechol in organic, nonpolar solvents to form thedesired catecholboranes. It was determined that the reaction sequenceshown in FIG. 15A worked: alkylcatecholborane 19 formed from freecatechol and an alkylboroinic acid in situ, and reacted withprofluorescent nitroxide 16 in one pot to form 22 with a stronglyfluorescent signal (FIG. 15B). This was an unexpectedly superior result.

FIG. 26 shows a successful field test of this detection system. Thecomposition was applied onto the surface of poison oak leaves. A papertowel was applied to the surface of the leaves and the paper towel wasilluminated using a UV-lamp. As shown in FIG. 26, the fluorescence wasclearly visible to the naked eye.

It has also been observed that the reaction works well in a variety ofpolar and nonpolar solvents.

Example III Synthesis and Development of the Components of theFluorescence-Generation Method: Optimize the Structure of the Nitroxide,Fluorophore, Tether and Alkylboronic Acid

The chemical design of the profluorescent nitroxide is explored,entailing the choice of the optimum nitroxide, fluorescent tag, andtether to prepare a robust, soluble and effective component for thisdetection system. As fluorescence is a very sensitive method ofdetection, only very small amounts need react to give a signal visibleto the naked eye using an inexpensive hand-held fluorescent lamp. Thesix-membered ring TEMPO is by far the most common nitroxide scaffold,however there are a number of other common stable nitroxide classes.Considerations in optimization of the nitroxide structure include easeand cost of synthesis, versatility in designing and optimizing thetether between the fluorophore and the nitroxide, stability andsolubility. Common stable nitroxide classes include TEMPO(tetramethylpiperidinyl-1-oxyl), proxyl (pyrrolidine analogues),nitronyl, imino and doxyl nitroxides (FIG. 16). The inventor and theinventor's research laboratory has been engaged in the synthesis andapplications of nitroxides for over a decade, thus has extensiveexperience in the synthesis of new nitroxides. In addition, a largenumber of commercially nitroxides are available from Toronto ResearchChemicals, Inc. (North York, Canada).

Recent work by Lozinsky et al. (2004) indicates that profluorescentnitronyl nitroxides quench fluorescence by a different mechanisminvolving nonbonding electrons of nitrogen and oxygen rather than to theunpaired electron. Thus the fluorescence does not increase uponreduction to the hydroxylamine (and also presumably from the formationof alkoxyamines), making them unsuitable for this study. Given thesimple synthetic access (FIG. 17) to proxyl nitroxides following thelarge body of work pioneered by Hideg, Keana, and many others, proxylnitroxides 42 are particularly attractive.

The fluorophore can be easily introduced late in the synthetic sequence,encouraging synthetic diversity without having to start the sequencefrom the beginning. For an example, a Grignard reagent 43 prepared from1-bromopyrene gives the proxyl nitroxide 44 with a very short tetherbetween the fluorophore and the nitroxide (FIG. 18).

Example IV Use of Fluorescence Detection

With regard to the choice of fluorophore, preliminary data and resultsfocused on Dansyl amino-TEMPO 12, a well-developed profluorescentnitroxide. One advantage of this compound is that sulfonamides areresistant to hydrolysis, thus minimizing the possibility of hydrolysisto give a free fluorophore and thus a false positive signal. Scaiano(Aliaga et al., Organic Lett., 2003, 5(22): 4145-4148) has developed4-(3-hydroxy-2-methyl-4-quinolinoyloxy)-TEMPO 45, which showssignificantly enhanced fluorescence upon reaction of the nitroxidecompared to Dansyl amino-TEMPO 12 (but contains a more easily hydrolyzedester linkage) (FIG. 19). Bottle (Micallef et al., Polymer Degrad.Stabil., 2005, 89(3): 427-435) has developed the profluorescentnitroxide TMDBIO 46, containing a phenanthrene fluorophore covalentlyfused into the structure of the nitroxide, making hydrolysis animpossibility. Other fluorophores such as pyrene 47 and coumarins havebeen utilized, and many more are possible. The use of fluorophoresobservable in the visible range is also explored. The intensity,wavelength dependence, cost, stability and ease of synthesis will all betaken into consideration in selecting the best fluorophore.

Efficient quenching requires a short tether between the fluorophore andthe nitroxide moiety; rotational freedom and flexibility also influencethe quenching efficiency. Thus the 5-membered ring nitroxides mayprovide an advantage in holding the fluorophore in a closer geometry tothe nitroxide as compared to the 6-membered ring framework of TEMPO.

Example V Quenching of Fluorophore

Dansyl amino TEMPO 12 does show a small amount of residual fluorescence,as shown in FIG. 20. Other profluorescent nitroxides may be even moreeffective at quenching the fluorescence in the free nitroxide state. Thewavelength of excitation and emission can be tuned by selection of thefluorophore.

Example VI Effect of Charge Upon Fluororescence Detection

Since urushiol is very hydrophobic, apolar organic solvents areinvestigated for the key reaction sequence, including toluene, hexanes,acetone, ethers, etc. The linear hydrophobic “tail” is optimized forboth reactivity with catechol and solubility to match that of thehydrophobic urushiol. B-alkylpinacolboranes 24 are conveniently preparedby iridium-catalyzed hydroboration⁷⁸ of the corresponding terminalalkenes using commercially available pinacolborane 23 (FIG. 21).Hydrolysis provides easy access to alkylboronic acids with a variety ofchain lengths. Commercially available C₁₂-C₁₇ linear terminal olefinsare available, with the C₁₄ and C₁₆ being particularly inexpensive. Upontesting with actual urushiol, there may be an advantage to having an oddor even number of carbons in the sidechain, or the exact carbon countmay prove to be inconsequential. The stability of the boronic acid isalso a consideration. Tertiary alkyl boronic acids are prone todecomposition upon exposure with air. In our preliminary studies, wehave used primary n-butyl boronic acid. The sample has remained stablefor over a year without taking any precautions to avoid exposure to air.We have determined that aryl boronic acids (very stable, andcommercially available) do not take part in the radical reactionsequence, presumably due to failure of the fragmentation step due to theinstability of aryl radicals. Thus primary alkyl boronic acids seem tobe ideal: they react in the desired radical reaction sequence, but arestable to storage.

Example VII Optimizing the Detection System with Regard toStoichiometry, Solvent, Concentration, Reaction Time, and Avoidance ofFalse Positives

Calibration of the fluorescence signal as a function of theconcentration of the catechol, boron reagent and nitroxide is carriedout. As exposure to 0.001 mg of urushiol can elicit allergic contactdermatitis, very small amounts of urushiol should to be detectable tomake this method effective. The optimal stoichiometry to obtain a shortreaction time is studied. It is expected that two nitroxides are neededfor every boron complex, although one equivalent may be sufficient ifthe nitroxide catecholboronate complex is hydrolytically unstable. Ifthe fluorescent signal is extremely strong, it may be possible toeconomize by using a mixture of regular nitroxide mixed with some smallpercentage of profluorescent nitroxide.

The specificity of this system for catechols is explored. As controls,phenols, resorcinols (1,3-benenediols), alcohols and diols (for example,sugars) are not expected to participate in the key reaction sequence, asno delocalized perboryl radical intermediate similar to 6 will beformed. Reaction with these various alcohols are tested to ensure thatthis method is indeed selective for catechols. Pyrogallols(1,2,3-benenetriols, for example gallocatechins (ex. 48) andepigallocatechins (FIG. 22) found in red wine, tea and chocolate) areexpected to participate in the reaction, depending upon their solubilityin the solvents. Likewise, the closely related catechins (ex. 49) andepicatechins (found in foods along with gallocatechins) are truecatechols: reactions are again be limited by solubility.

Possible sources of false positives are examined. It is well known thatnitroxides react rapidly with ascorbic acid to form hydroxylamines. Ourresearch group has used ascorbate reduction of nitroxide to aid inchromatographic separation of alkoxyamine from unreacted nitroxide.Blough was the first to show profluorescent nitroxides react withascorbic acid to generate a fluorescent signal. Lozinsky has utilizedprofluorescent nitroxides to assay the amount of vitamin C in fruitjuices, and Wang has used a fluorescent conductive charged polymernitroxide salt as a sensor for ascorbate and for trolox (a vitamin Emimic) Another side reaction that may interfere with the selectivedetection of urushiol by this boron catechol sequence is the simplereduction of nitroxides by phenols. Scaiano has studied the kinetics ofhydrogen transfer from phenol to nitroxide using a profluorescentnitroxide. The rate constants are very slow: k=0.003 M⁻¹s⁻¹ in proticsolvent for gallic acid and BHT, and k=0.2 M⁻¹s⁻¹ for TROLOX. Scaianodid not investigate reduction by catechol. In preliminary experiments(FIG. 23), we have shown that addition of catechol to Dansyl amino-TEMPO12 in toluene does produce a weak fluorescent signal, however this issuppressed by addition of a mild oxidant (PbO₂) to convert the tinyamount of hydroxylamine to nitroxide. This removes the false positivefrom phenol (FIG. 24).

The use of other mild oxidants that will rapidly oxidize hydroxylamineto nitroxide in organic solvents, but not oxidize catechol to quinone,are investigated (See FIG. 25). Particularly attractive are Fe^((III))salts as less toxic alternatives to lead. We have determined that OXONEis too strong of an oxidizing agent: the nitroxide is oxidized to theoxammonium salt. Interestingly, Bottle has shown that pyrrolidinenitroxides (cyclic 5-membered rings) have higher reduction potentialsthan piperidine (6-membered ring) nitroxides. Thus use of a pyrrolidineprofluorescent nitroxide may inhibit the false positive signal arisingfrom reduction by phenols.

REFERENCES

Addition of Nitroxides to Catecholboranes:

-   Schaffner and Renaud (2004) Eur. J. Org. Chem. 2291-2298.-   Darmency and Renaud, (2006) Top. Curr. Chem. 263: 71-106.-   Cadot et al., (2002) J. Org. Chem., 67; 7193-7202.-   Ollivert et al. (1999) Synlett. 6: 807-809.

Attempted Addition of Nitroxides to Trialkylboranes:

-   Braslau and Anderson, in Radicals in Organic Synthesis, vol. 2    (Eds. P. Renaud, M. P. Sibi), Wiley-VCH, Weinheim, 2001, p. 129.

Addition of Oxygen Radicals to Catecholboranes:

-   Baban et al. (1986) J. Chem. Soc., Perkin Trans 2: 157.-   Suzuki et al. (1969) J. Chem. Soc., Chem. Commun 1009.-   Brown and Negishi (1971) J. Am. Chem. Soc. 93: 3777.-   Forster (1999) PhD Thesis, Université de Fribourg, Switzerland,    Diss. Nr. 1242.-   Ollivier and Renaud (1999) Chem. Eur. J. 5: 1468.-   Kumli et al. (2006) Organic Lett. 8(25): 5861-5864.-   Ollivier and Renaud (2000) Angew. Chem. Int. Ed. Eng. 39: 925.

Profluorescent Nitroxides:

-   Blough (1988) J. Am. Chem. Soc. 110: 1915.-   Blough (1990) Free Rad. Res. Comm 10: 119-121.-   Blough (1996) Anal. Chem. 68: 867-872.-   Micallef A S et al. (2005) Polym Degrad. & Stability 89: 427-435.-   Foitzik et al. (2008) Macromolecules 41: 1577-1580.-   Blinco et al. E. J. Org. Chem. 28: 4638-4641.-   Sciano (2001) Macromol. 34: 6184.-   Coenjarts et al. (2003) J. Am. Chem. Soc 125: 620-621.-   Ivan et al. (2003) Photochem. Photobiol. 78: 416.-   Aspee et al. (2007) Photochem. Photobiol. 83(3): 481-485.-   Maurel et al. (2006) J. Phys. Chem. B, 110(33): 16353-16358.-   Laferriere et al. (2006) Chem. Comm (3): 257-259.-   Aspee et al. (2003) Photochem. Photobiol. Sci. 2(11): 1125-1129.-   Aspee et al. (2003) Macromolecules, 36(10): 3550-3556.-   Korth (2000) Biol. Chem. 381(7): 575-582; ibid (1999) Chem. Eur. J.    5(6): 1738-1747;-   ibid (1997) Angew. Chem. Int. Ed. Eng. 36: 1501-1503; ibid (2006)    Helv. Chim Acta 89: 2281-2296.-   Zhang and Zhu (2004) Chem. Commun 670.-   Hideg (2006) Photochem. Photobiol. 82: 1211.-   Wang (2006) Chem. Mater. 18: 3605-3610.-   Dang and Guo (2006) Appl. Spectrosc. 60: 203-207.-   Likhtenstein et al. (2007) Photochem. Photobiol. 83: 871-881.-   Lozinsky, et al. (2004) Anal. Biochem. 326: 139-145.-   Likhtenstein (2002) Biochem. Biophys. Meth. 51: 1-15.-   Likhtenstein (1990) Analyst 115: 839.-   Likhtenstein (1999) Biochem. Biophys. Meth. 38: 29-42.

Those skilled in the art will appreciate that various adaptations andmodifications of the just-described embodiments can be configuredwithout departing from the scope and spirit of the invention. Othersuitable techniques and methods known in the art can be applied innumerous specific modalities by one skilled in the art and in light ofthe description of the present invention described herein. Therefore, itis to be understood that the invention can be practiced other than asspecifically described herein. The above description is intended to beillustrative, and not restrictive. Many other embodiments will beapparent to those of skill in the art upon reviewing the abovedescription. The scope of the invention should, therefore, be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

I claim:
 1. A kit for detecting a catechol, the kit comprising acompound, an applicator, a vessel, the vessel shaped and adapted forconfining the compound, the compound comprising a nitroxide and aneffective amount of a boron compound, the boron compound comprising analkylboronic acid having the general formula R—B(OH)₂.
 2. The kit ofclaim 1, wherein the boron compound comprises a hydrophobic alkyl group.3. The kit of claim 1, wherein the boron compound comprises ahydrophilic alkyl group.
 4. The kit of claim 1, wherein the nitroxide isa profluorescent nitroxide.
 5. The kit of claim 1, wherein theapplicator is a spray applicator.
 6. The kit of claim 1, wherein thecatechol is urushiol.
 7. The kit of claim 1 further comprising anaerosol propellant.
 8. The kit of claim 1 further comprising a lamp. 9.The kit of claim 1, wherein the boron compound optionally comprises atleast one B-alkyl boronic acid derivative.
 10. The kit of claim 1wherein the boron compound is present in an amount selected from thegroup consisting of from about 99.5% to about 0.001%, from about 95% toabout 0.1%, and from about 90% to about 0.5%, by weight, based on thetotal combined weight of the boron composition thereof.
 11. The kit ofclaim 1, wherein the compound comprises the boron compound in aneffective amount for the detection of a catechol in poison oak oil. 12.A kit for detecting a catechol, the kit comprising a compound, anapplicator, a vessel, the vessel shaped and adapted for confining thecompound, the compound comprising a nitroxide and a boron compound, theboron compound comprising an alkylboronic acid having the generalformula R—B(OH)₂, where R is selected from the group consisting of ahydrophobic alkyl group and a hydrophilic alkyl group.
 13. The kit ofclaim 12, wherein the nitroxide is a profluorescent nitroxide.
 14. Thekit of claim 12, wherein the applicator is a spray applicator.
 15. Thekit of claim 12, wherein the catechol is urushiol.
 16. The kit of claim12 further comprising an aerosol propellant.
 17. The kit of claim 12further comprising a lamp.
 18. The kit of claim 12, wherein the boroncompound optionally comprises at least one B-alkyl boronic acidderivative.
 19. The kit of claim 12 wherein the boron compound ispresent in an amount selected from the group consisting of from about99.5% to about 0.001%, from about 95% to about 0.1%, and from about 90%to about 0.5%, by weight, based on the total combined weight of theboron composition thereof.
 20. The kit of claim 12, wherein the compoundcomprises the boron compound in an effective amount for the detection ofa catechol in poison oak oil.